Methods and Compositions for the Prevention of and Treatment of Infections Utilizing Chitosan-Derivative Compounds

ABSTRACT

The present invention is directed to the treatment and prevention of nosocomial infections or MRSA infections utilizing soluble chitosan or chitosan derivative compounds. These chitosan-derivative compounds, e.g., chitosan-arginine and chitosan-acid amines, exhibit bactericidal activity against bacterial pathogens, e.g., drug resistant bacteria such as Methicillin-resistant  Staphylococcus aureus  (MRSA).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/189,149 filed onAug. 16, 2008, and U.S. Ser. No. 61/158,328 filed on Mar. 6, 2009. Thecontents of the aforementioned applications are hereby incorporated byreference in their entirety.

GOVERNMENT SUPPORT

As outlined under 37 CFR 401.14(b), the United States government shallhave a nonexclusive, nontransferable, irrevocable, paid-up license topractice or have practiced for or on behalf of the United States thesubject invention.

FIELD OF THE INVENTION

The invention relates to soluble chitosan and derivatized chitosan andtheir use to treat bacterial infections, e.g., nosocomial infections orMRSA infections.

BACKGROUND

The prevalence and virility of nosocomial infections, coupled with thegrowing threat of antibiotic resistance and side effects of certainmedications, highlight the urgent need for improved methods to safelyand effectively prevent increasingly common multi-drug resistantinfections. Medical facilities have reported increasing rates ofnosocomial infections with major complications of burn injuries due tobacterial infections. Likewise, rising multi-drug resistance increasesthe risk and magnitude of infection in traumatic and surgical wounds. Inthe civilian setting in 2007, approximately 1.7 million people acquire anosocomial infection while hospitalized, resulting in nearly 100,000deaths annually. The majority of these infections are attributed tomethicillin-resistant Staphylococcus aureus (MRSA). While hospitals haveinstituted improved hygiene habits for caregivers as well as MRSAadmission screening protocols, these implementations have noteffectively served to reduce the spread of nosocomial MRSA. In additionto the hospital environment, the close quarters of prisons and daycarecenters may also facilitate elevated rates of nosocomial infections. Theneed for safe, prophylactic infection prevention is imperative to helpcontrol nosocomial infections and save lives. A rapid, cost-effectiveand nontoxic method of decontamination offers the greatest potential toreduce nosocomial infection. The Food and Drug Administration hasrecognized the bactericidal activity of chitosan. Research by our grouphas demonstrated an extremely broad-spectrum antimicrobial capability ofchitosan-based materials, which indicates the potential to createnon-toxic, efficacious, wide-ranging technologies to kill multi-drugresistant microorganisms in a variety of applications. Of keyimportance, because they are a fibrous polysaccharide that is notabsorbed or metabolized by the body, these chitosan-based products arebiocompatible.

Chitosan, a biopolymer derived from the second most abundant polymer onearth, chitin, has been widely used as a hemostatic bandage, cellularscaffold, and dietary supplement because it is biocompatible,biodegradable, and non-toxic. Chitosan has also shown antibacterialproperties only present when dissolved in aqueous acidic solutions. Inacid, the chitosan become polycationic, a property associated both withits solubility in acid as well as its antibacterial properties. We havedeveloped chitosan based derivatives that readily dissolve in neutralwater or saline which demonstrate enhanced antibacterial propertieswhile maintaining the desirable characteristics of chitosan mentionedpreviously. These compounds will not require the area to be sealedduring treatment and pose no threat to human health or the environment.In addition, the material can be stored as a dry powder or dissolved inwater, saline, or other neutral solution with a long shelf life anddispersed when needed.

Multi-drug resistant Enterococci, Staphylococci and Pneumococci havebecome common nosocomial pathogens. These drug resistant organisms havebeen rapidly spreading by horizontal transfer of resistance elements andclonal spread through the medical system. Because these resistantorganisms also form intractable biofilms on tissue and medical devices,deaths from these infections have been rising throughout the past twodecades. More recently, community acquired methicillin resistantStaphylococcus aureus (CA-MRSA) have been associated with increasinglyvirulent infections, including toxic shock syndrome (TSS), purpurafulminans and necrotizing fasciitis. Recent work with CA-MRSA strainshas implicated Panton-Valentin leukocidin (PVL) as a major virulencedeterminant. See M. Labandiera-Rey et al., “Staphylococcus AureusPanton-Valentine Leukocidin Causes Necrotizing Pneumonia,” 315 Science,11130 (2007); B. Said-Salim et al., “Differential Distribution AndExpression of Panton-Valentine Leucocidin Among Community-AcquiredMethicillin-Resistant Staphylococcus Aureus Strains,” 43 J. Clin.Microbiol, 3373 (2005); J. Voyich et al., “Is Panton-ValentineLeukocidin The Major Virulence Determinant In Community-AssociatedMethicillin-resistant Staphylococcus Aureus Disease?” 194 J. Infect.Dis., 1761 (2006); and Loren G. Miller et al., “Necrotizing FasciitisCaused by Community-Associated Methicillin-Resistant StaphylococcusAureus in Los Angeles,” 352 New England J. of Med., 1445, No. 14 (2005).Additionally, because of the rapid onset of these toxicoses, the use oftraditional systemic antibiotics is frequently unhelpful.

Chitosan and chitosan derivative compounds demonstrate effectivetreatment of MRSA. Because chitosan is biodegradable, non-toxic andexhibits antibacterial activity against a broad spectrum ofmicroorganisms, it has been utilized in the medical arena. However, theuse of chitosan is limited because of its insolubility at neutral andphysiological pH.

SUMMARY OF THE INVENTION

Methods to treat or prevent bacterial infection, e.g., nosocomialinfections or MRSA infections using soluble chitosans or derivatizedchitosans are described herein.

It is an objective of the present invention to provide a biocompatiblesoluble chitosanchitosan-derivative compound that is exhibit broadspectrum antimicrobial abilities.

It is an objective of the present invention to provide a chitosanderivative compound that is shelf-stable in dry powder form or dissolvein water, saline, or other neutral solution and dispersed as needed.

It is also an objective of the present invention to provide a solublechitosan or chitosan-derivative compound that is capable of treatingnosocomial infections or MRSA infection.

It is also an objective of the present invention to provide achitosan-derivative compound that is capable of preventing the spread ofnosocomial infections or MRSA infection.

It is also an objective of the present invention to provide achitosan-derivative compound that is effective at preventing growth ofMRSA on surfaces.

It is also an objective of the present invention to provide achitosan-derivative compound that shows potent efficacy as abactericidal in multiple Staphylococcus aureus strains.

It is also an objective of the present invention to provide achitosan-derivative compound that is capable of controlling the growthof MRSA by clumping.

These and other objectives are described herein.

In one aspect, the invention features a method of treating or preventinga nosocomial infection (or disorder), Staphylococcus infection (ordisorder), or methicillin-resistant Staphylococcus aureus (MRSA)infection (or disorder), reducing or preventing the spread of nosocomialinfection, Staphylococcus infection (or disorder), or MRSA infection,reducing Staphylococcus or MRSA load, or treating or preventing asymptom of nosocomial infection, Staphylococcus infection (or disorder),or MRSA infection, the method comprises:

administering, to a subject who has nosocomial infection, Staphylococcusinfection (or disorder), or MRSA infection, or a symptom of nosocomialinfection, Staphylococcus infection (or disorder), or MRSA infection, oris at risk of nosocomial infection, Staphylococcus infection, or MRSAinfection, an effective amount of soluble chitosan or derivatizedchitosan, thereby treating or preventing the nosocomial infection (ordisorder), Staphylococcus infection (or disorder), or MRSA infection (ordisorder), reducing or preventing the spread of nosocomial infection,Staphylococcus infection, or MRSA infection, reducing Staphylococcus orMRSA load, or treating or preventing the symptom of nosocomialinfection, Staphylococcus infection, or MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of Staphylococcus infection (ordisorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of Staphylococcus infection (ordisorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce Staphylococcus load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce MRSA load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of MRSA infection.

In some embodiments, the subject is a human or an animal, e.g., a horseor a cow.

In some embodiments, the nosocomial infection, Staphylococcus infection,or MRSA infection (or disorder) has previously been treated with anantibiotic without a soluble chitosan or derivatized chitosan, e.g.,said treatment was unsatisfactory.

In some embodiments, the subject has one or more symptoms selected froma group consisting of: a red, swollen and painful area on the skin,drainage of pus or other fluids from the infected site, fever, skinabscess, warmth around the infected area, chest pain, chills, fatigue,fever, malaise, headache, muscle aches, rash, wound, skin breach, and/orshortness of breath.

In some embodiments, the subject is at risk for nosocomial infection(disorder) or MRSA infection (or disorder), e.g., a person with weakimmune system (e.g., an AIDS patient, a cancer patient, or a severeasthmatic), a diabetic, a cystic fibrosis patient, an athleteparticipating in contact sports or weight training, a young children, anelderly, a person staying in a health care facility for an extendedperiod of time, a person living in confined space with other people, aperson using an invasive devise (e.g., a person who is on dialysis, iscatheterized, or has feeding tubes), a person who has recent antibioticuse (e.g., treatment with fluoroquinolones (Ciprofloxacin, Ofloxacin orLevofloxacin) or cephalosporin antibiotics in the past four weeks), or asurfer who spend large amounts of time in coast waters where MRSA ispresent.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered topically, enterally or parenterally.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered by inhalation (e.g., nasal) spray.

In some embodiments, the effective amount is therapeutically effectiveamount.

In some embodiments, the soluble chitosan or derivatized chitosan is notadministered in combination with a second therapy, e.g., an antibiotic,(e.g., Clindamycin, Linezolid, Tetracycline,Trimethoprim-sulfamethoxazole, or Vancomycin).

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of Staphylococcus spread in a subject by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to asubject who has not been treated with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces Staphylococcus load in the subject by at least 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to theStaphylococcus load in the subject before treatment with the solublechitosan or derivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of MRSA spread in a subject by at least 10, 20, 30,40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to a subjectwho has not been treated with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces MRSA load in the subject by at least 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 99, 99.9, or 99.99%, compared to the MRSA load in thesubject before treatment with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino that is substituted with a nitrogenprotecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

In another aspect, the invention features a pharmaceutical compositionfor treating and preventing a nosocomial infection (or disorder),Staphylococcus infection (or disorder), or MRSA infection (or disorder),reducing or preventing the spread of a nosocomial infection,Staphylococcus infection, or MRSA infection, reducing, Staphylococcus orMRSA load, or treating or preventing a symptom of a nosocomialinfection, Staphylococcus infection, or MRSA infection in a subject,wherein the composition comprises a soluble chitosan or derivatizedchitosan described herein.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce Staphylococcus load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce MRSA load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of MRSA infection.

In some embodiments, the subject is a human or an animal, e.g., a horseor a cow.

In some embodiments, the nosocomial infection, Staphylococcus infection,or MRSA infection (or disorder) has previously been treated with anantibiotic without a soluble chitosan or derivatized chitosan, e.g.,said treatment was unsatisfactory.

In some embodiments, the subject has one or more symptoms selected froma group consisting of: a red, swollen and painful area on the skin,drainage of pus or other fluids from the infected site, fever, skinabscess, warmth around the infected area, chest pain, chills, fatigue,fever, malaise, headache, muscle aches, rash, wound, skin breach, and/orshortness of breath.

In some embodiments, the subject is at risk for nosocomial infection(disorder), Staphylococcus infection (disorder), or MRSA infection (ordisorder), e.g., a person with weak immune system (e.g., an AIDSpatient, a cancer patient, or a severe asthmatic), a diabetic, a cysticfibrosis patient, an athlete participating in contact sports or weighttraining, a young children, an elderly, a person staying in a healthcare facility for an extended period of time, a person living inconfined space with other people, a person using an invasive devise(e.g., a person who is on dialysis, is catheterized, or has feedingtubes), a person who has recent antibiotic use (e.g., treatment withfluoroquinolones (Ciprofloxacin, Ofloxacin or Levofloxacin) orcephalosporin antibiotics in the past four weeks), or a surfer who spendlarge amounts of time in coast waters where MRSA is present.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered topically, enterally or parenterally.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered by inhalation (e.g., nasal) spray.

In some embodiments, the effective amount is therapeutically effectiveamount.

In some embodiments, the soluble chitosan or derivatized chitosan is notadministered in combination with a second therapy, e.g., an antibiotic,(e.g., Clindamycin, Linezolid, Tetracycline,Trimethoprim-sulfamethoxazole, or vancomycin).

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of Staphylococcus spread in a subject by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to asubject who has not been treated with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces Staphylococcus load in the subject by at least 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to theStaphylococcus load in the subject before treatment with the solublechitosan or derivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of MRSA spread in a subject by at least 10, 20, 30,40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to a subjectwho has not been treated with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces MRSA load in the subject by at least 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 99, 99.9, or 99.99%, compared to the MRSA load in thesubject before treatment with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

The composition of claim x, wherein R³ is C₂ alkyl substituted with anamino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino that is substituted with a nitrogenprotecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

In yet another aspect, the invention features a kit for treating andpreventing of a nosocomial infection (or disorder) or MRSA infection (ordisorder), reducing or preventing the spread of a nosocomial infectionor MRSA infection, reducing MRSA load, or treating or preventing asymptom of a nosocomial infection or MRSA infection in a subject,wherein the composition comprises a soluble chitosan or derivatizedchitosan described herein.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent nosocomial infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent Staphylococcus infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent MRSA infection (or disorder).

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent the spread of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce Staphylococcus load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to reduce MRSA load in the subject.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of nosocomial infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of Staphylococcus infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to treat a symptom of MRSA infection.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered to prevent a symptom of MRSA infection.

In some embodiments, the subject is a human or an animal, e.g., a horseor a sow.

In some embodiments, the nosocomial infection (or disorder),Staphylococcus infection (or disorder), or MRSA infection (or disorder)has previously been treated with an antibiotic without a solublechitosan or derivatized chitosan, e.g., said treatment wasunsatisfactory.

In some embodiments, the subject has one or more symptoms selected froma group consisting of: a red, swollen and painful area on the skin,drainage of pus or other fluids from the infected site, fever, skinabscess, warmth around the infected area, chest pain, chills, fatigue,fever, malaise, headache, muscle aches, rash, wound, skin breach, and/orshortness of breath.

In some embodiments, the subject is at risk for nosocomial infection (ordisorder), Staphylococcus infection (or disorder), or MRSA infection (ordisorder), e.g., a person with weak immune system (e.g., an AIDSpatient, a cancer patient, or a severe asthmatic), a diabetic, a cysticfibrosis patient, an athlete participating in contact sports or weighttraining, a young children, an elderly, a person staying in a healthcare facility for an extended period of time, a person living inconfined space with other people, a person using an invasive devise(e.g., a person who is on dialysis, is catheterized, or has feedingtubes), a person who has recent antibiotic use (e.g., treatment withfluoroquinolones (Ciprofloxacin, Ofloxacin or Levofloxacin) orcephalosporin antibiotics in the past four weeks), or a surfer who spendlarge amounts of time in coast waters where MRSA is present.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered topically, enterally or parenterally.

In some embodiments, the soluble chitosan or derivatized chitosan isadministered by inhalation (e.g., nasal) spray.

In some embodiments, the effective amount is therapeutically effectiveamount.

In some embodiments, the soluble chitosan or derivatized chitosan is notadministered in combination with a second therapy, e.g., an antibiotic,(e.g., Clindamycin, Linezolid, Tetracycline,Trimethoprim-sulfamethoxazole, or Vancomycin).

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of Staphylococcus spread in a subject by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to asubject who has not been treated with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces Staphylococcus load in the subject by at least 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to theStaphylococcus load in the subject before treatment with the solublechitosan or derivatized chitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the chance of MRSA spread in a subject by at least 10, 20, 30,40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to a subjectwho has not been treated with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan or derivatized chitosanreduces MRSA load in the subject by at least 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 99, 99.9, or 99.99%, compared to the MRSA load in thesubject before treatment with the soluble chitosan or derivatizedchitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino that is substituted with a nitrogenprotecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

In one aspect, the invention features a method of inhibiting the growth(e.g., killing) of a bacterium (e.g., MRSA) in a sample, comprisingcontacting the sample with an effective amount of soluble chitosan orderivatized chitosan, thereby inhibiting the growth (e.g., killing) ofthe bacterium (e.g., MRSA) in said sample.

In some embodiments, the sample is a clinical (e.g., hospital) sample,e.g., whole blood, plasma, serum, urine, saliva, stool, sweat, tears,stool.

In some embodiments, the bacterium is a bacterium isolated from aclinical sample, e.g., a bacterium described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the growth rate of the bacterium (e.g., MRSA) by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to abacteria that has not been contacted with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

The method of any one of claims x-x, wherein R² is amino that issubstituted with a nitrogen protecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

In another aspect, the invention features a composition for inhibitingthe growth (e.g., killing) of a bacterium (e.g., MRSA) in a sample,comprising contacting the sample with an effective amount of solublechitosan or derivatized chitosan described herein.

In some embodiments, the sample is a clinical (e.g., hospital) sample,e.g., whole blood, plasma, serum, urine, saliva, stool, sweat, tears, orstool.

In some embodiments, the bacterium is a bacterium isolated from aclinical sample, e.g., a bacterium described herein.

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the growth rate of the bacterium (e.g., MRSA) by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to abacteria that has not been contacted with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino that is substituted with a nitrogenprotecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

In yet another aspect, the invention features a kit for inhibiting thegrowth (e.g., killing) of a bacterium (e.g., MRSA) in a sample,comprising contacting the sample with an effective amount of solublechitosan or derivatized chitosan.

In some embodiments, the sample is a clinical (e.g., hospital) sample,e.g., whole blood, plasma, serum, urine, saliva, stool, sweat, tears,stool.

In some embodiments, the bacterium is a bacterium isolated from aclinical sample, e.g., a bacterium described herein.

In some embodiments, the MRSA is selected from EMRSA15 strain, EMRSA16strain (ST36:USA200 or MRSA252), CC8 strain designated ST8:USA300,ST8:USA400 strain, ST8:USA500 strain, ST59:USA1000 strain, ST59 strain,ST80 strain, ST93 strain, MW-2 strain, MNHO strain, clinical isolatesfrom a hospital, and other MRSA strains described herein.

In some embodiments, the bacterium is selected from other Staphylococcusspecies such as S. aureus, S. epidermidis, S. staprophyticus, S.lugdunensis, S. schleiferi, and S. caprae and their drug resistantstrains, clinical isolates from a hospital, and/or other Staphylococcusstrains described herein.

In some embodiments, the soluble chitosan or derivatized chitosanreduces the growth rate of the bacterium (e.g., MRSA) by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to abacteria that has not been contacted with the soluble chitosan orderivatized chitosan.

In some embodiments, the soluble chitosan is soluble in aqueous solutionbetween pH 6.8 and pH 7.4.

In some embodiments, the soluble chitosan has a molecule weight lessthan 10,000 kDa.

In some embodiments, the derivatized chitosan comprises a chitosan ofthe following formula (I):

wherein:

n is an integer between 20 and 6000; and

each R¹ is independently selected for each occurrence from hydrogen,acetyl, and a group of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety,

wherein R² is hydrogen or amino; and

R³ is amino, guanidino, C₁-C₆ alkyl substituted with an amino orguanidino moiety, or a natural or unnatural amino acid side chain,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof formula (II).

In some embodiments, between 25-95% of R¹ substituents are hydrogen.

In some embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl.

In some embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group offormula (II).

In some embodiments, between 4-30% of R¹ substituents are a group offormula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

AND at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino that is substituted with a nitrogenprotecting group.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc). For example, in some embodiments, in thesynthetic process a nitrogen protecting group is used, which can providean intermediate polymer having a nitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.

In some embodiments, R³ is C₁ alkyl substituted with an amino group.

In some embodiments, R³ is C₂ alkyl substituted with an amino group.

In some embodiments, R³ is C₃ alkyl substituted with an amino group.

In some embodiments, R³ is C₄ alkyl substituted with an amino group.

In some embodiments, R³ is C₅ alkyl substituted with an amino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup.

In some embodiments, R³ is C₁ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₂ alkyl substituted with a guanidino group.

In some embodiments, R³ is C₃ alkyl substituted with a guanidino group.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituentsindependently selected from any of the formulae specifically shownabove.

In some embodiments, the derivatized chitosan of formula (I) may befurther derivatized on the free hydroxyl moieties.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 1,000,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 350,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 60,000 Da.

In some embodiments, the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.

In some embodiments, the derivatized chitosan is soluble in aqueoussolution between pH 6.8 and pH 7.4.

In some embodiments, the derivatized chitosan is substantially free ofother impurities.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows planktonic killing of Staphylococcus aureus.

FIG. 1A shows the demonstrated bacteriocide of S. aureus ATCC strain29213 at 1 hour.

FIG. 1B shows the demonstrated bacteriocide of S. aureus ATCC strain29213 at 20 hours.

FIG. 1C shows the demonstrated bacteriocide of S. aureus ATCC strain25923 at 1 hour.

FIG. 1D shows the demonstrated bacteriocide of S. aureus ATCC strain25923 at 20 hours.

FIG. 2 shows reduction of S. aureus in clinical isolates in 0.1M acetatebuffer at pH 7. FIG. 2A shows demonstrated bacteriocide of MN8 at 1hour.

FIG. 2B shows demonstrated bacteriocide of MN8 at 20 hours.

FIG. 2C shows demonstrated bacteriocide of MNHO at 1 hour.

FIG. 2D shows demonstrated bacteriocide of MNHO at 20 hours.

FIG. 2E shows demonstrated bacteriocide of MNDO at 1 hour.

FIG. 2F shows demonstrated bacteriocide of MNDO at 20 hours.

FIG. 2G shows demonstrated bacteriocide of MW-2 at 1 hour.

FIG. 2H shows demonstrated bacteriocide of MW-2 at 20 hours.

FIG. 3 shows chitosan-arginine's ability to kill MRSA isolates.

FIG. 4 shows the prevention of bacterial and biofilm growth on surfaces.

FIG. 5 shows biofilm reduction accomplished by the addition ofchitosan-arginine.

FIG. 6 shows that chitosan-arginine causes the clumping of bacteria.

FIG. 6A shows images of the bacteria in water alone as a control.

FIG. 6B shows images of the bacteria treated for 1 minute, at 400×magnification.

FIG. 7 shows the mechanism of bactericide is membrane disruption.

FIG. 8 shows a preliminary study to examine MRSA MW-2's ability todevelop resistance to chitosan-arginine.

FIG. 9 shows a comparison between chitosan-acid amine andchitosan-arginine against MW-2.

FIG. 10 shows chitosan-acid amine's ability to clump MRSA.

FIG. 10A shows an image of the bacteria in water alone as a control.

FIG. 10B shows an image of the bacteria treated for 1 minute, at 200×magnification.

FIG. 11 shows a comparison of the antibacterial activity of chitosanderivatives against MRSA.

FIGS. 12A and 12B show the antibacterial activity of chitosan-arginineagainst MRSA in human saliva.

FIG. 13 shows the ability of various chitosan lots to clump MRSA.

DETAILED DESCRIPTION

Described herein are methods and compositions that are useful fortreating or preventing bacterial infections, e.g., nosocomial infectionsor MRSA infections. Exemplary methods generally include use of solublechitosans or deriviatized chitosans. In some embodiments, the solublechitosans or derivatized chitosans exhibit one or more of the followingcharacteristics: for example, long shelf lives, ability to be stored asa dry powder, or ability to dissolve in water, saline, or other neutralsolution and to be dispersed as needed.

The compounds described herein can be used to treat or prevent bacterialinfections, e.g., nosocomial infections or MRSA infections. Exemplarycompounds include, but not limited to soluble chitosan compounds,chitosan-arginine compounds, chitosan-guanidine compounds,chitosan-unnatural amino acid compounds, chitosan-acid-amine compounds,chitosan-natural amino acid compounds, co-derivatives of the justdescribed compounds and the salts thereof. These compounds and theirantimicrobial activity are disclosed in U.S. patent application Ser. No.11/657,382, which is herein incorporated by reference.

The compounds described herein can be effective in the treatment andprevention of the spread of nosocomial infections or MRSA infections.

The compounds described herein can be used to treat or prevent bacterialinfection, e.g., gram-positive or gram-negative bacteria. These bacteriacan be across different phases of growth, e.g., biofilms attached to twodifferent abiotic surfaces.

The compounds described herein can also be more favorable in treatmentand prevention of infections associated with resistant bacteria, e.g.,MRSA (e.g., HA-MRSA (e.g., strain MNHO) and CA-MRSA (e.g., strainMW-2)).

DEFINITIONS

As used herein nosocomial infections refers to infection which is aresult of treatment in a hospital or a healthcare service unit, butsecondary to the patient's original condition. Infections are considerednosocomial if they first appear 48 hours or more after hospitaladmission or within 30 days after discharge. This type of infection isalso known as a hospital-acquired infection (or more genericallyhealthcare-associated infections).

As used herein resistant microorganism or bacterium means, an organismwhich has become resistant to an anti-bacterial agent. In embodiments athe minimum inhibitory concentration of a resistant bacterium will be atleast, 2, 5, 10, or 100 greater than for that seen with a non-resistantbacterium for a selected anti-bacterial agent.

As used herein primary contact means that an individual is in directphysical contact with the subject or that they exchange bodily fluids,e.g., by drinking from the same cup. Secondary contact means that afirst individual has primary contact with a second individual and thesecond individual has direct contact with the subject.

Treatment

The compounds described herein (e.g., a soluble chitosan or aderivatized chitosan) can be administered to cells in culture, e.g. invitro or ex vivo, or to a subject, e.g., in vivo, to treat, prevent,and/or diagnose a variety of disorders, including those described hereinbelow.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a compound (e.g., a compound describedherein (e.g., a soluble chitosan or a derivatized chitosan) to asubject, e.g., a patient, or application or administration of thecompound to an isolated tissue or cell, e.g., cell line, from a subject,e.g., a patient, who has a disorder (e.g., a disorder as describedherein), a symptom of a disorder, or a predisposition toward a disorder,with the purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve or affect the disorder, one or more symptoms of thedisorder or the predisposition toward the disorder (e.g., to prevent atleast one symptom of the disorder or to delay onset of at least onesymptom of the disorder).

As used herein, the term “prevent” or “prevention” is defined as theapplication or administration of a compound (e.g., a compound describedherein (e.g., a soluble chitosan or a derivatized chitosan)) to asubject, e.g., a subject who is at risk for a disorder (e.g., a disorderdescribed herein), or has a disposition toward a disorder, orapplication or administration of the compound to an isolated tissue orcell, e.g., cell line, from a subject, e.g., a subject who is at riskfor a disorder (e.g., a disorder as described herein), or has apredisposition toward a disorder, with the purpose to avoid or precludethe disorder, or affect the predisposition toward the disorder (e.g., toprevent at least one symptom of the disorder or to delay onset of atleast one symptom of the disorder).

As used herein, an amount of a compound effective to treat a disorder,or a “therapeutically effective amount” refers to an amount of thecompound which is effective, upon single or multiple dose administrationto a subject, in treating a cell, or in curing, alleviating, relievingor improving a subject with a disorder beyond that expected in theabsence of such treatment.

As used herein, an amount of a compound effective to prevent a disorder,or “a prophylactically effective amount” of the compound refers to anamount effective, upon single- or multiple-dose administration to thesubject, in preventing or delaying the occurrence of the onset orrecurrence of a disorder or a symptom of the disorder.

As used herein, the term “subject” is intended to include human andnon-human animals. Exemplary human subjects include a human patienthaving a disorder, e.g., a disorder described herein or a normalsubject. The term “non-human animals” of the invention includes allvertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles)and mammals, such as non-human primates, domesticated and/oragriculturally useful animals, e.g., sheep, dog, cat, cow, pig, etc.

As used herein, “administered in combination” or a combinedadministration of two agents means that two or more agents (e.g.,compounds described herein) are administered to a subject at the sametime or within an interval such that there is overlap of an effect ofeach agent on the patient. Preferably they are administered within 15,10, 5, or 1 minute of one another. Preferably the administrations of theagents are spaced sufficiently close together such that a combinatorial(e.g., a synergistic) effect is achieved. The agents can be administeredsimultaneously, for example in a combined unit dose (providingsimultaneous delivery of both agents). Alternatively, the agents can beadministered at a specified time interval, for example, an interval ofminutes, hours, days or weeks. Generally, the agents are concurrentlybioavailable, e.g., detectable, in the subject.

In a preferred embodiment, the agents are administered essentiallysimultaneously, for example two unit dosages administered at the sametime, or a combined unit dosage of the two agents. In another preferredembodiment, the agents are delivered in separate unit dosages. Theagents can be administered in any order, or as one or more preparationsthat includes two or more agents. In a preferred embodiment, at leastone administration of one of the agents, e.g., the first agent, is madewithin minutes, one, two, three, or four hours, or even within one ortwo days of the other agent, e.g., the second agent. In some cases,combinations can achieve synergistic results, e.g., greater thanadditive results, e.g., at least 20, 50, 70, or 100% greater thanadditive.

Functionalized Chitosan Derivatives

Methods, compounds and compositions for treating or preventing bacterialinfections, e.g., nosocomial infections or MRSA infections, aredescribed herein.

Chitosan is derived from chitin, which is a polymer ofN-acetylglucosamine that is the main component of the exoskeletons ofcrustaceans (e.g. shrimp, crab, lobster). Chitosan is formed from chitinby deacetylation, and as such is not a single polymeric molecule, but aclass of molecules having different molecular weights and differentdegrees of deacetylation. The percent deacetylation in commercialchitosans is typically between 50-100%. The chitosan derivativesdescribed herein are generated by functionalizing the resulting freeamino groups with positively charged moieties, as described herein. Thederivatized chitosans described herein have a number of properties whichare advantageous for a nucleic acid delivery vehicle including: theyeffectively bind and complex the negatively charged nucleic acids, theycan be formed into nanoparticles of a controllable size, they be takenup by the cells and they can release the nucleic acids at theappropriate time within the cell.

Chitosans with any degree of deacetylation greater than 50% are used inthe present invention, with functionalization between 2% and 50%.(Percent functionalization is determined relative to the number of freeamino moieties on the chitosan polymer.) The degrees of deacetylationand functionalization impart a specific charge density to thefunctionalized chitosan derivative. The resulting charge density affectssolubility, nucleic acid binding and subsequent release, and interactionwith mammalian cell membranes. Thus, in accordance with the presentinvention, these properties must be optimized for optimal efficacy.Exemplary chitosan derivatives are described in Baker et al; Ser. No.11/657,382 filed on Jan. 24, 2007, which is incorporated herein byreference.

The chitosan derivatives described herein have a range of molecularweights that are soluble at neutral and physiological pH, and includefor the purposes of this invention molecular weights ranging from5-1,000 kDa. Embodiments described herein are feature lower molecularweight of derivatized chitosans (<25 kDa, e.g., from about 5 to about25) which can have desirable delivery and transfection properties, andare small in size and have favorable solubilities. A low molecularweight derivatized chitosan is generally more soluble than a highermolecular weight, the former thus producing a nucleic acid/chitosancomplex that will release the nucleic acid and provide increasedtransfection of cells. Much literature has been devoted to theoptimization of all of these parameters for chitosan based deliverysystems.

The functionalized chitosan derivatives described herein include thefollowing:

(A) Chitosan-arginine compounds;

(B) Chitosan-natural amino acid derivative compounds:

(C) Chitosan-unnatural amino acid compounds;

(D) Chitosan-acid amine compounds; and

(E) Chitosan-guanidine compounds.

(F) Neutral chitosan derivative compounds.

(A) Chitosan-Arginine Compounds

In some embodiments, the present invention is directed tochitosan-arginine compounds, where the arginine is bound through apeptide (amide) bond via its carbonyl to the primary amine on theglucosamines of chitosan:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

or a racemic mixture thereof,

wherein at least 25% of R¹ substituents are H, at least 1% are acetyl,and at least 2% are a group of the formula shown above.

(B) Chitosan-Natural Amino Acid Derivative Compounds

In some embodiments, the present invention is directed tochitosan-natural amino acid derivative compounds, wherein the naturalamino acid may be histidine or lysine. The amino is bound through apeptide (amide) bond via its carbonyl to the primary amine on theglucosamines of chitosan:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

or a racemic mixture thereof, wherein at least 25% of R¹ substituentsare H, at least 1% are acetyl, and at least 2% are a group of theformula shown above; OR a group of the following formula:

or a racemic mixture thereof, wherein at least 25% of R¹ substituentsare H, at least 1% are acetyl, and at least 2% are a group of theformula shown above.

(C) Chitosan-Unnatural Amino Acid Compounds

In some embodiments, the present invention is directed tochitosan-unnatural amino acid compounds, where the unnatural amino acidis bound through a peptide (amide) bond via its carbonyl to the primaryamine on the glucosamines of chitosan:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

wherein R³ is an unnatural amino acid side chain, and wherein at least25% of R¹ substituents are H, at least 1% are acetyl, and at least 2%are a group of the formula shown above.

Unnatural amino acids are those with side chains not normally found inbiological systems, such as ornithine (2,5-diaminopentanoic acid). Anyunnatural amino acid may be used in accordance with the invention. Insome embodiments, the unnatural amino acids coupled to chitosan have thefollowing formulae:

(D) Chitosan-Acid Amine and Guanidine Compounds

In some embodiments, the present invention is directed to chitosan-acidamine compounds, or their guanidylated counterparts. The acid amine isbound through a peptide (amide) bond via its carbonyl to the primaryamine on the glucosamines of chitosan:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

wherein R³ is selected from amino, guanidino, and C₁-C₆ alkylsubstituted with an amino or a guanidino group, wherein at least 25% ofR¹ substituents are H, at least 1% are acetyl, and at least 2% are agroup of the formula shown above

In some embodiments, R¹ is selected from one of the following:

(E) Chitosan-Guanidine Compounds

In some embodiments, the present invention is directed tochitosan-guanidine compounds.

wherein each R¹ is independently selected from hydrogen, acetyl, and ortogether with the nitrogen to which it is attached, forms a guanidinemoiety; wherein at least 25% of R¹ substituents are H, at least 1% areacetyl, and at least 2% are a group of the formula shown above.

(F) Neutral Chitosan Derivative Compounds

In some embodiments, the present invention is directed to neutralchitosan derivative compounds. Exemplary neutral chitosan derivativecompounds include those where one or more amine nitrogens of thechitosan has been covalently attached to a neutral moiety such as asugar:

wherein each R¹ is independently selected from hydrogen, acetyl, and asugar (e.g., a naturally occurring or modified sugar) or an α-hydroxyacid. Sugars can be monosaccharides, disaccharides or polysaccharidessuch as glucose, mannose, lactose, maltose, cellubiose, sucrose,amylose, glycogen, cellulose, gluconate, or pyruvate. Sugars can becovalently attached via a apacer or via the carboxylic acid, ketone oraldehyde group of the terminal sugar. Examples of α-hydroxy acidsinclude glycolic acid, lactic acid, and citric acid. In some preferredembodiments, the neutral chitosan derivative is chitosan-lactobionicacid compound or chitosan-glycolic acid compound. Exemplary salts andcoderivatives include those known in the art, for example, thosedescribed in US 20070281904, the contents of which is incorporated byreference in its entirety.

Bacterial Pathogens

Methods described herein are useful for inhibiting the growth ofbacteria, e.g., bacteria that cause nosocomial infections (e.g., MRSA,such as CA-MRSA and HA-MRSA), and bacteria that are resistant toantibiotics (e.g., MRSA, such as CA-MRSA and HA-MRSA).

Exemplary nosocomial pathogens include, e.g., commensal bacteria foundin normal flora of healthy humans (e.g., cutaneous coagulase negativestaphylococci in intravascular line infection, and intestinalEscherichia coli in urinary infection), and pathogenic bacteria havinggreater virulence and causing infections (sporadic or epidemic)regardless of host status (e.g., Anaerobic Gram-positive rods (e.g.Clostridium), Gram-positive bacteria (e.g., Staphylococcus aureus, andbeta-haemolytic streptococci), Gram-negative bacteria (e.g.,Enterobacteriacae (e.g. Escherichia coli, Proteus, Klebsiella,Enterobacter, Serratia marcescens), and Pseudomonas spp.), and otherbacteria (e.g., Legionella species).

Exemplary pathogens that cause resistant bacterial infections include,e.g., Methicillin resistant Staphylococcus aureus, Fluoroquinoloneresistant Staphylococcus aureus, Vancomycin intermediate resistantStaphylococcus aureus, Linezolid resistant Staphylococcus aureus,Penicillin resistant Streptococcus pneumoniae, Macrolide resistantStreptococcus pneumoniae, Fluorocμiinolone resistant Streptococcuspneumoniae. Vancomycin resistant Enterococcus faecalis, Linezolidresistant Enterococcus faecalis, Fluoroquinolone resistant Enterococcusfaecalis, Vancomycin resistant Enterococcus faecium, Linezolid resistantEnterococcus faecium. Fluoroquinolone resistant Enterococcus faecium,Ampicillin resistant Enterococcus faecium, Macrolide resistantHaemophilus influenzae, β-lactam resistant Haemophilus influenzae,Fluoroquinolone resistant Haemophilus influenzae, β-lactam resistantMoraxella catarrhalis, Methicillin resistant Staphylococcus epidermidis,Methicillin resistant Staphylococcus epidermidis. Vancomycin resistantStaphylococcus epidermidis, Fluoroquinolone resistant Staphylococcusepidermidis, Macrolide resistant Mycoplasma pneumoniae, Isoniazidresistant Mycobacterium tuberculosis, Rifampin resistant Mycobacteriumtuberculosis, Methicillin resistant Coagutase negative Staphylococci,Fluoroquinolone resistant Coagulase negative Staphylococci, Glycopeptideintermediate resistant Staphylococcus aureus, Vancomycin resistantStaphylococcus aureus, Hetero vancomycin intermediate resistantStaphylococcus aureus, Hetero vancomycin resistant Staphylococcusaureus, Macrolide-Lincosamide-Streptogramin resistant Staphylococcus,β-lactam resistant Enterococcus faecalis, β-lactam resistantEnterococcus faecium, Ketolide resistant Streptococcus pneumoniae,Ketolide resistant Streptococcus pyogenes, Macrolide resistantStreptococcus pyogenes, or Vancomycin resistant Staphylococcusepidermidis.

Subjects

The subject can be a human or an animal. Exemplary disorders in humansand animals include: those described above, for example as caused by aninfection with a bacterium described above. Suitable animal subjectsinclude: but are not limited to, pet, wild, zoo, laboratory, and farmanimals. Suitable animal subjects include primates, rodents, and birds.Examples of said animals include, but not limited to, guinea pigs,hamsters, gerbils, rat, mice, rabbits, dogs, cats, horses, pigs, sheep,cows, goats, deer, rhesus monkeys, monkeys, tamarinds, apes, baboons,gorillas, chimpanzees, orangutans, gibbons, fowl, e.g., pheasant, quail(or other gamebirds), a waterfowl, ostriches, chickens, turkeys, ducks,and geese or free flying bird.

Exemplary disorders in humans include diseases characterized by thepresence of one or more of the bacteria that are associated withnosocomial infections, e.g., commensal bacteria found in normal flora ofhealthy humans (e.g., cutaneous coagulase negative staphylococci inintravascular line infection, and intestinal Escherichia coli in urinaryinfection), and pathogenic bacteria having greater virulence and causinginfections (sporadic or epidemic) regardless of host status (e.g.,Anaerobic Gram-positive rods (e.g. Clostridium), Gram-positive bacteria(e.g., Staphylococcus aureus, and beta-haemolytic Streptococci),Gram-negative bacteria (e.g., Enterobacteriacae (e.g. Escherichia coli,Proteus, Klebsiella, Enterobacter, Serratia marcescens), and Pseudomonasspp.), and other bacteria (e.g., Legionella species).

Exemplary disorders in humans and animals also include diseasescharacterized by the presence of one or more of the bacteria that causeresistant bacterial infections such as Methicillin resistantStaphylococcus aureus, Fluoroquinolone resistant Staphylococcus aureus,Vancomycin intermediate resistant Staphylococcus aureus, Linezolidresistant Staphylococcus aureus, Penicillin resistant Streptococcuspneumoniae, Macrolide resistant Streptococcus pneumoniae,Fluorocμiinolone resistant Streptococcus pneumoniae. Vancomycinresistant Enterococcus faecalis, Linezolid resistant Enterococcusfaecalis, Fluoroquinolone resistant Enterococcus faecalis, Vancomycinresistant Enterococcus faecium, Linezolid resistant Enterococcusfaecium. Fluoroquinolone resistant Enterococcus faecium, Ampicillinresistant Enterococcus faecium, Macrolide resistant Haemophilusinfluenzae, β-lactam resistant Haemophilus influenzae, Fluoroquinoloneresistant Haemophilus influenzae, β-lactam resistant Moraxellacatarrhalis, Methicillin resistant Staphylococcus epidermidis,Methicillin resistant Staphylococcus epidermidis. Vancomycin resistantStaphylococcus epidermidis, Fluoroquinolone resistant Staphylococcusepidermidis, Macrolide resistant Mycoplasma pneumoniae, Isoniazidresistant Mycobacterium tuberculosis, Rifampin resistant Mycobacteriumtuberculosis, Methicillin resistant Coagutase negative Staphylococci,Fluoroquinolone resistant Coagulase negative Staphylococci, Glycopeptideintermediate resistant Staphylococcus aureus, Vancomycin resistantStaphylococcus aureus, Hetero vancomycin intermediate resistantStaphylococcus aureus, Hetero vancomycin resistant Staphylococcusaureus, Macrolide-Lincosamide-Streptogramin resistant Staphylococcus,β-lactam resistant Enterococcus faecalis, β-lactam resistantEnterococcus faecium, Ketolide resistant Streptococcus pneumoniae,Ketolide resistant Streptococcus pyogenes, Macrolide resistantStreptococcus pyogenes, or Vancomycin resistant Staphylococcusepidermidis.

In some embodiments, the subject has stye, sinusitis, boils, carbuncles,furuncles, hematogenous spread, endocarditis, pneumonia, emesis,impetigo, diarrhea, scaled skin syndrome, toxic shock syndrome, urinarycystitis, and/or ostenmyelitis.

Formulations and Routes of Administration

The compounds described herein can be formulated in a variety ofmanners, including for oral, topical or systemic delivery (e.g.,administered orally, parenterally, by inhalation spray (e.g., nasalspray), nebulizer, topically, rectally, nasally, buccally). In someembodiments, inhalation sprays (e.g., nasal spray or nasal mists), areused for the nasal delivery of a compound descried herein, eitherlocally or systemically to treat or prevent an infection or disorderdescribed herein, e.g., a nosocomial infection or MRSA infection.Inclusion in feed, water or an inhaled formulation is particularlydesirable for use with animals.

The compounds described herein (e.g., a soluble chitosan or aderivatized chitosan) can, for example, be administered by injection,intravenously, intraarterially, subdermally, intraperitoneally,intramuscularly, or subcutaneously; or orally, buccally, nasally,transmucosally, topically, in an ophthalmic preparation, or byinhalation, with a dosage ranging from about 0.001 to about 100 mg/kg ofbody weight, e.g., between 0.001-1 mg/kg, 1-100 mg/kg, or 0.01-5 mg/kg,every 4 to 120 hours, e.g., about every 6, 8, 12, 24, 48, or 72 hours,or according to the requirements of the particular compound. Thecompound described herein can be administered before or after the onsetof the disorder described herein. The methods herein contemplateadministration of an effective amount of compound or compoundcomposition to achieve the desired or stated effect. Typically, thepharmaceutical compositions of this invention will be administered fromabout 1 to about 6 times per day. Alternatively, the compounds can beadministered as a continuous infusion. Such administration can be usedas a chronic or acute therapy. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage formwill vary depending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;an additional compound including for example, a steroid or an analgesic;and any pharmaceutically acceptable carrier, adjuvant or vehicle.Alternate compositions of this invention comprise a compound describedherein or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, adjuvant or vehicle. Thecompositions delineated herein include the compounds described herein,as well as additional therapeutic compounds If present, in amountseffective for achieving a modulation of disease or disease symptoms.

The compositions are generally made by methods including the steps ofcombining a compound described herein with one or more carriers and,optionally, one or more additional therapeutic compounds delineatedherein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase which can be combined with emulsifying and/orsuspending agents. If desired, certain sweetening and/or flavoringand/or coloring agents may be added.

The compounds of this invention may be administered by aerosol,nebulizer, or inhalation. In some embodiments, the composition is in theform of a dry powder, a suspension, or a solution. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art. Exemplary methods and devices for aerosol orinhalation include those described in U.S. Pat. No. 6,962,151, which isincorporated herein by reference in its entirety.

Compositions formulated for inhaled delivery generally include particleshaving a mean diameter of from about 0.1 μm to about 50 μm (e.g., fromabout 0.1 μm to about 10 μm, or from about 0.2 μm to about 5 μm. In someembodiments, the composition includes a dispersion of suitably-sized dryparticles, for example, precipitants or crystals) or a dispersion of asolution (e.g., droplets) of a suitable size.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein.

In some cases, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form.

The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination ofcompounds described herein, both the compounds are generally present atdosage levels of between about 1 to 100%, and more preferably betweenabout 5 to 95% of the dosage normally administered in a monotherapyregimen. Additionally, combinations of a plurality of compoundsdescribed herein are also envisioned. The compounds may be administeredseparately, as part of a multiple dose regimen, from the compounds ofthis invention. Alternatively, those compounds may be part of a singledosage form, mixed together with the compounds of this invention in asingle composition.

Kits

A compound described herein (e.g., a soluble chitosan or a derivatizedchitosan (can be provided in a kit. The kit includes (a) a compositionthat includes a compound described herein, and, optionally (b)informational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of the compound described herein for themethods described herein.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates touse of the compound described herein to treat a disorder describedherein.

In one embodiment, the informational material can include instructionsto administer the compound described herein in a suitable manner toperform the methods described herein, e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). Preferred doses, dosage forms, ormodes of administration are parenteral, e.g., intravenous,intramuscular, subcutaneous, intraparenteral, bucosal, sublingual,intraoccular, and topical. In another embodiment, the informationalmaterial can include instructions to administer the compound describedherein to a suitable subject, e.g., a human, e.g., a human having or atrisk for a disorder described herein. For example, the material caninclude instructions to administer the compound described herein to sucha subject.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to a compound described herein, the composition of the kitcan include other ingredients, such as a solvent or buffer, astabilizer, a preservative, and/or a second compound for treating acondition or disorder described herein. Alternatively, the otheringredients can be included in the kit, but in different compositions orcontainers than the compound described herein. In such embodiments, thekit can include instructions for admixing the compound described hereinand the other ingredients, or for using a compound described hereintogether with the other ingredients.

The compound described herein can be provided in any form, e.g., liquid,dried or lyophilized form. It is preferred that the compound describedherein be substantially pure and/or sterile. When the compound describedherein is provided in a liquid solution, the liquid solution preferablyis an aqueous solution, with a sterile aqueous solution being preferred.When the compound described herein is provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent, e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the compositioncontaining the compound described herein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of acompound described herein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of a compound described herein. The containers of thekits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, pipette, forceps, measuredspoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or woodenswab), or any such delivery device.

In a preferred embodiment, the device is an implantable delivery device.

EXAMPLES

DDA=degree of deacetylation PDI=polydispersity

Example 1 Staphylococcus aureus from Culture Strains, Isolates andClinical Isolates is Killed by Chitosan Derivatives

Bacterial cultures were maintained as frozen stocks in a −80° freezeruntil use. The culture was thawed, and inoculated from the stock bulletat a 1:100 dilution into fresh sterile Todd Hewitt broth. The culturegrew at 37° C. with shaking for 18 h in a G-76 water bath incubator.Culture OD (595 nm) is measured and bacteria were pelleted. The cellswere resuspended at OD595 0.05 in 100 mM sodium acetate at pH 7.0, and10-fold dilutions of the culture were pipetted in 100 μl aliquots into asterile, clear polystyrene 96-well round bottom microtiter dish. After 1h of incubation at room temperature, the culture was washed withphosphate buffered saline, washed again with Todd-Hewitt broth andresuspended in the same volume of broth Microtiter plates werecentrifuged and culture fluid was removed gently with a multichannelpipetter. The plate was prepared as described previously by Brewster(2003). The prepared microtiter calibration dish was loaded into amicroplate reader, and incubated at 37° C. for 18 h. Readings were takenat 595 nm with a 15 s agitation prior to each reading. The dilutionswere plated on Todd-Hewitt agar to determine a corresponding growth ofcolony forming units for the inoculum. After the completion of thegrowth curve experiments, growth rates were calculated. The error forthese calculations, determined as the mean value±SE, was always lowerthan the instrument error of 0.003 absorbance units. The time tothreshold value for each dilution was plotted against initial cellconcentration to determine a calibration curve. In accordance with thepresent invention, chitosan arginine is shown to provide bactericidalactivity.

FIG. 1 shows planktonic killing of Staphylococcus aureus, ATCC strainsin 0.1M acetate buffer at pH 7. Bacteria were incubated for 1 h (blackbars) or 20 h (white bars). Bacteria were all in stationary phase ofculture when tested. Data shows surviving virtual colony forming units(vCFU's) as described in the text for doses from 25 to 200 μg/ml for 51kDa with 4% functionalization (% DDA 83, PDI 2.05); and 35.2 kDa 30%functionalized (% DDA 83) chitosan-arginine. Error is CFU+/−SE based onplate counting performed both with growth curve calibration and tests.Demonstrated bacteriocide of S. aureus ATCC strains 29213 at 1 hour(FIG. 1A) and at 20 hours (FIG. 1B) and of S. aureus ATCC 25923 at 1hour (FIG. 1C) and at 20 hours (FIG. 1D). Note that after 20 h thelevels of ATCC 25923 were below the detection limit of the assay withall doses tested (theoretically 10 cfu/ml). The strain showed sporadicgrowth in the microtiter wells.

FIG. 2 shows reduction of S. aureus in clinical isolates in 0.1M acecatebuffer at pH 7. FIG. 2A shows demonstrated bacteriocide of MN8 at 1hour, FIG. 2B shows demonstrated bacteriocide of MN8 at 20 hours. FIG.2C shows demonstrated bacteriocide of MNHO at 1 hour and FIG. 2D showsdemonstrated bacteriocide of MNHO at 20 hours. FIG. 2E showsdemonstrated bacteriocide of MNDO at 1 hour and FIG. 2F showsdemonstrated bacteriocide of MNDO at 20 hours. FIG. 2G showsdemonstrated bacteriocide of MW-2 at 1 hour and FIG. 2H showsdemonstrated bacteriocide of MW-2 at 20 hours. After 20 h exposure, MNDOand MN8 levels were below the assay's theoretical detection limit of 10cfu/ml. Inferred surviving cfu below this line are reported as 10cfu/ml. Data shows surviving virtual colony forming units (vCFU's) asdescribed in the text for doses from 25 to 200 μg/ml for 51 kDa with 4%functionalization (% DDA 83, PDI 2.05); and 35.2 kDa 30% functionalized(% DDA 83, PDI 1.71) chitosan-arginine. Bacteria were all in stationaryphase of culture when tested. Error is CFU+/−SE based on plate countingperformed both with growth curve calibration and tests. Bacteria areexplained in Table 1.

FIGS. 1-2 show planktonic exposure of Staphylococcus aureus tochitosan-arginine. The bacterial culture strains utilized include (ATCC)type strain 25923, (ATCC) strain, 29213, pathogenic Staphylococcusaureus strains MN8, a vaginal toxic shock syndrome (TSS) isolate, TSST-1producer, (MNDO), a wound isolate that produces SEC, (MNHO) anmethicillin resistant Staphylococcus aureus (MRSA(70)), a wound isolateand community-acquired methicillin-resistant Staphylococcus aureus(CA-MRSA) strain MW2. ATCC type strain 25923 was used for preliminaryexperiments to show susceptibility. The strains are listed in Table 1,below.

TABLE 1 Bacterial species and strain Importance Source S. aureus ATCC25923 Initial test strain Carolina Biologicals S. aureus ATCC 29213Clinical isolate S. aureus MW-2 CA-MRSA P.M. Schlievert S. aureus MNHOWound Isolate P.M. Schlievert (MRSA, SEB) S. aureus MNDO Wound Isolate(SEC) P.M. Schlievert S. aureus MN8 TSS isolate P.M. Schlievert E.faecalis Endocarditis, VRE Carolina Biologicals Str. pyogenesNecrotizing fasciitis Carolina Biologicals Str. pneumoniae Meningitis,pneumonia Carolina Biologicals

FIG. 3 shows twelve strains of clinical isolates were acquired fromProvidence Medical Center (Portland, Oreg.) to test the ability ofchitosan-arginine to kill a variety of MRSA clinical isolates. The MRSAwere isolated from various sites of infection/colonization and included3 each from the skin (1-3), respiratory tract/sputum (4-6), blood (7-9),and nasal orifice (10-12). A single concentration of chitosan-arginine(100 μg/mL) 28% functionalized and 23.8 kDa (% DDA 83, PDI 1.54) wasused to treat approximately 10⁶ CFU/mL for 24 hours at room temperature.This dose was chosen because it is the lowest dose used for consistentand significant reductions of MRSA MW-2 in previous studies. Followingtreatment the cells were centrifuged and resuspended in water beforebeing diluted and plated for CFU on Todd-Hewitt Agar plates. The CFU/mLmeasured are reported for each of the 12 isolates. Note thatchitosan-arginine sterilized the strains as the lack of a grey bar is anindication of less than 10CFU counted.

As shown in FIGS. 1-3, chitosan-arginine is highly bactericidal againstall ATCC strains, four clinical isolates tested, including the wellcharacterized community acquired MRSA strain MW-2 and hospital acquiredstrains. A dose and time response was observed.

Example 2 Chitosan-Arginine as a Spray Prevents MRSA Growth

FIG. 4 shows the prevention of bacterial and biofilm growth on surfaces.Studies were completed to assess the response of methicillin-resistantstaphylococcus aureus (MRSA) to a surface coating of chitosan-arginine.Chitosan-arginine 28% functionalized with 23.8 kDa (% DDA 83, PDI 1.54)in a solution of 90% ethanol in water was deposited with varying surfacecoverage to polystyrene surfaces (12-well plate) and allowed to drycompletely overnight. Subsequently, 1×10⁶ bacteria (200 μL of 5×10⁶bacteria/mL) of and MRSA MW-2 were deposited on the surface andrecovered after 4 hour or 24 hours. The surviving bacteria were platedand counted. Note that even at very low surface coverage (<10 μg/cm²),nearly complete sterilization MRSA is observed for these large bacterialchallenges. FIG. 4 shows a dose response of MRSA MW-2 to a pretreatmentof very low amounts of chiotsan-arginine on a surface. This prophylacticuse of chiotsan-arginine is effective at preventing the growth of MRSAtransferred to a surface.

Example 3 Chitosan-Arginine Reduces MRSA in Preformed Biofilms

The activity of chitosan-arginine (35.2 kDa, 30% functionalized, % DDA83, PDI

1.71) against MW-2 biofilms was assessed using a CDC biofilm reactor.The biofilms were grown in polycarbonate wells. After 3 d of growth in astirred, aerated batch culture, the biofilms were removed and treatedwith 100 μg/ml chitosan arginine in water. Alternatively, untreatedsamples were incubated alongside treated samples in water. All growthand treatment took place at room temperature. After exposure, sampleswere removed from treatment, and evaluated by plate counting andviability staining as described above. The plate counts wereaccomplished by scraping the outer surface of the coupons with a flatend stock stick. After thorough scraping, the stick was placed in 500 μlsterile Millipore water. The samples were diluted in phosphate bufferedsaline, plated on agar, and counted for growth of colony forming units.The susceptibility of MW-2 biofilms to treatment with chitosan-arginineprovided dramatic results.

As shown in FIG. 5, biofilm reduction is accomplished by the addition ofchitosan arginine. Chitosan-arginine was used to reduce bacteria inpreformed biofilms. MW-2 biofilms were grown in Todd Hewitt broth for2.5 days. The biofilm were washed and treated with 0, 10, 50 or 100μg.mL of chitosan-arginine 28% functionalized, 23.8 kDa (% DDA 83, PDI1.54) for 4 hours. The biofilms were scraped from the wells, diluted andplated to count for colony forming units, CFU. The data shows theremaining CFU after this exposure and shows up to a four log reductionin bacteria.

Example 4 Chitosan-Arginine Clumps Staphylococcus aureus and CausesMembrane Permeability

The methicilin resistant strain MW-2 is rapidly clumped by the additionof chitosan-arginine in solution as shown in FIG. 6. FIG. 6 shows thatchitosan-arginine causes the clumping of bacteria. This physicalinteraction is hypothesized to facilitate aggregation andpermabilization of the bacterial cell membrane. To test this hypothesis,planktonic MW-2 was treated for 1-minute with 100 μg/mlchitosan-arginine 28% functionalized, 23.8 kDa (% DDA 83, PDI 1.54) andbacteria were stained with a BacLight Live/Dead staining kit (MolecularProbes) to observe cell aggregation and determine the extent of membranepermeability. A red signal indicates that bacterial membranepermeability has increased, allowing propidium iodide to intercolateinto double stranded DNA as opposed to cells labeled with the green DNAdye. The bacteria were imaged in water alone as a control as shown inFIG. 6A. FIG. 6B shows images of the bacteria or treated for 1-minute,in 400× magnification. As can be seen from the figures, this clumpingoccurs in less than 1 minute, which is the time limit to make themeasurement.

Furthermore, the bacteria become permeable, and ATP is measured outsidethe cell in as short as 15 minutes, the limit of our detection time forthis assay, as shown in FIG. 7. The process of killing is slow, however,relative to the leakage of adenosine triphosphate (ATP). Membranepermeability was determined by measuring the amount of ATP secreted intothe supernatant of a bacterial preparation of MW-2 exposed to variousdoses of chitosan-arginine in water with 37% functionalized, 40 kDa (%DDA 89, PDI 2.45). In this luminescence assay, ATP is measured as afunction of time. FIG. 7 shows that at very low doses, the membraneleaks ATP, and that at longer time periods, more ATP has leaked out,suggesting that the time course of permeability and killing is not aninstant bactericidal process.

Example 5 Lack of Resistance by MRSA to the Chitosan-DerivativeCompounds

In accordance with the present invention staphylococci are unable todevelop resistance against these chitosan derivative compounds asdetermined via a serial exposure and recovery experiment. When the fourisolates (MW2, MNDO, MNHO, MN8) and MNHO were challenged repeatedly withchitosan-arginine (10 passages) and compared to a control that waspassed repeatedly without challenge, the bactericidal efficacy of 100μg/ml chitosan-arginine (4% and 30%) remained unchanged. FIG. 8 is apreliminary study to examine the ability of MRSA MW-2 to developresistance to chitosan-arginine demonstrated no resistance development.The study is first divided into three groups indicated by the color ofthe bar to provide repetitive treatment and re-growth of surviving MRSA.Chitosan-arginine used was 51 kDa with 4% functionalization (% DDA 83,PDI 2.05) and 35.2 kDa 30% functionalized (% DDA 83). The bacteria weretreated for 1-hour with either 100 mM acetate buffer alone at pH 7(white, control), with 4% (grey) or with 30% (black) functionalizedchitosan-arginine. The surviving bacteria, with a doubling time of about30 min, were resuspended and grown to full culture over 18 hours. Thetreatment, rinsing and regrowth was repeated ten times in the samemanner with the same treatment, so that in each iteration only thesurviving bacteria were recultured, and again, challenged with the samechitosan-arginine or control. Upon completion of these ten repetitivechallenges, three isolates from each of the control, 4% and 30%functionalized chitosan-arginine challenges were isolated, cultured andsubjected to a final 20-hour treatment with buffer (nochitosan-arginine) in group A, 4% in group B and 30% functionalizedchitosan-arginine in group C. The resulting surviving colonies wereenumerated to determine if the multiple challenges reduced bacterial(MRSA) susceptibility to chitosan-arginine. Note that all survivingbacteria from the various treatments grew normally when exposed tobuffer (as shown in A). All surviving bacteria from the varioustreatments were equally inhibited by the 4% in group B and all survivingbacteria from the various treatments were inhibited by the 30% in groupC, and within the error, equally so. These data suggest that thesusceptibility of MW-2 is unchanged by this level of repetitive exposureto chitosan-arginine.

As shown in FIG. 8, no alteration of susceptibility in strain MW-2 wasobserved, although the viability of pretreated cells under non-growthconditions (in sodium acetate buffer) was altered by serial exposure.All four clinical isolates were reduced to below the assay detectionlimit after 20 h exposure to 100 μg/ml irrespective of pretreatment (notshown).

Example 6 Chitosan-Acid Amine has Bactericidal and Clumping ActivityAgainst MRSA

Chitosan-derivatives such as the 6 amino-hexanoic acid derivative ofchitosan, also show activity against MRSA. Other chitosan derivativesare active against MW-2 as well. A bacterial preparation of MW-2 wasexposed to 100 mg/mL of chitosan-arginine (28% functionalized, 23.8 kDa,% DDA 83, PDI 1.54) and chitosan-acid amine (38% functionalized, 28.7kDa, % DDA 83, PDI 1.47), 6 amino hexanoic acid) for 24 hours andvirtual colony forming units as described (vCFU) were determined. Whileboth reduce the MW-2 viability, the acid-amine derivative of chitosanfor this particular strain of bacteria is less effective, but stillreduces the bacterial density by three logs

In FIG. 9, chitosan-acid amine is compared to chitosan-arginine againstMW-2. Although its activity is not as great for planktonic growth asthis particular chitosan-arginine, its MW and % functionalization havenot been optimized for MW-2. Furthermore, chitosan-acid amine alsoclumps MW-2 as shown in FIG. 10.

FIG. 10 shows Chitosan-acid amine's ability to clump MRSA. In thisexample, planktonic MW-2 was treated for 1-minute with 100 μg/mlchitosan-acid amine (38% functionalized, 28.7 kDa, % DDA 83, PDI 1.47)and bacteria were stained with a BacLight Live/Dead staining kit(Molecular Probes) to observe cell aggregation and determine the extentof membrane permeability. A red signal indicates that bacterial membranepermeability has increased, allowing propidium iodide to intercolateinto double stranded DNA as opposed to cells labeled with the green DNAdye. The bacteria were imaged in water alone as a control as shown inFIG. 10A or treated for 1-minute, as shown in FIG. 10B and shown in 200×magnification. The positively charged derivatives of chitosan will havevariable activity against the variety of strains of MRSA and allow forthe control of MRSA populations by optimization of the derivativesrelative to the strain.

Example 7 Comparative Testing of the Antibacterial Activity of ChitosanDerivatives Against MRSA

The molecules comprise chitosan-arginine (C/A),chitosan-guanidinobutyric acid (C/A4G), and chitosan-aminocaproic acid(C/A6A). Standard carbodiimide coupling techniques were employed tosynthesize each derivative. Each of these molecules possesses apositively charged amino or guanidinium group when dissolved in anaqueous solution at pH 7 which allows the derivative to be soluble,unlike the non-water soluble polymer chitosan which requires acidconditions for solubility.

The chitosan derivatives were comparatively tested to determine theantibacterial activity of each against Methicillin ResistantStaphylococcus aureus (MRSA) strain MW-2. Note that the shorthandnotation of molecule such as C/A (L,H) refers to (% functionalization,MW) so that a C/A (L,H) has a low % functionalization and a highermolecular weight. The chitosan derivatives are listed in Table 2, below,each of which has DDA 89%.

TABLE 2 Chitosan-Arginine: 16%  36 kDa (PDI 2.10) C/A (L, L) 15% 103 kDa(PDI 1.44) C/A (L, H) 31%  52 kDa (PDI 1.35) C/A (H, L) 31%  73 kDa (PDI1.44) C/A (H, H) Chitosan-Aminocaproic Acid 12%  57 kDa (PDI 2.30) C/A6A(L, L) 12% 126 kDa (PDI 2.12) C/A6A (L, H) 34%  92 kDa (PDI 1.93) C/A6A(H, H) 34%  43 kDa (PDI 1.93) C/A6A (H, L) Chitosan-GuanidinobutyricAcid 12%  31 kDa (PDI 2.31) C/A4G (L, L) 13%  84 kDa (PDI N/A) C/A4G (L,H)

Planktonic bacteria were grown in the appropriate broth media for 18 hat 37° C., while shaking. Approximately 10⁶/ml bacteria were resuspendedin water and the chitosan derivatives were added at 100 μg/ml for 4hours to MRSA. These tests were completed in 96-well polystyrene platesin a volume of 200 μl. Following the treatment period the plates werecentrifuged, supernatants were aspirated, and bacteria resuspended inthe appropriate culture media and grown in the Envision 2104 multilabelreader (PerkinElmer) for 18 hours with the OD595 measure at 5 minuteintervals. The time to threshold 0.25 was calculated and used todetermine the vCFU following each treatment. The result is shown in FIG.11.

Example 8 Chitosan Arginine has Antibacterial Activity Against MRSA inHuman Saliva

Antibacterial activity of C/A (25% functionalization, 18 kDa, % DDA 88,PDI 1.47) against MRSA MW-2 was assessed in human saliva. The saliva wascollected from one healthy donor, centrifuged, and the supernatant wasstored on ice. Approximately 10⁷-10⁸/mL MRSA were suspended in thesaliva and incubated with C/A for 4 hours. The C/A concentrations usedwere; 0, 25, 50, and 100 μg/ml and were in a saliva solution. Resultsshowed that C/A in saliva maintains significant antimicrobial activity(FIGS. 12A and 12B).

Example 9 Testing for the Clumping Ability of Various Chitosan LotsAgainst MRSA

MRSA MW-2 (5 ml) was tested for the ability of various chitosan lots toaggregate the material in water or PBS. The % DDA for each of the C/Adescribed below is 88. C/A lots (BC04 41 kDa 26% functionalized (PDI1.88), BC07 34 kDa 10% functionalized (PDI 2.03), BC08 18 kDa 25%functionalized (PDI 1.47), BC09 28 kDa 25% functionalized (PDI 1.71),BC10 43kDa 25% functionalized (PDI 2.28)) were tested at a concentrationof 100 μg/ml. The derivatives were added to the bacterial suspensions in15 ml plastic test tubes and 3 samples from each in a volume of 100 μlwere obtained from the middle (to avoid misrepresentation of theobservation because of natural bacterial settling due to gravity). Adecrease in the OD 595 over time indicated the bacteria are aggregatingand falling out of the suspension. The result is shown in FIG. 13. MW-2aggregation was obvious visually after 24 hours and showed all lots wereaggregating MW-2. First, MW2 was not clumping in PBS with BC08 added.Further testing showed that it was able to clump under these conditions.

Other embodiments are within the following claims.

1. A method of treating or preventing a nosocomial infection,Staphylococcus infection, or methicillin-resistant Staphylococcus aureus(MRSA) infection reducing or preventing the spread of nosocomialinfection, Staphylococcus infection, or MRSA infection, reducingStaphylococcus or MRSA load, or treating or preventing a symptom ofnosocomial infection, Staphylococcus infection, or MRSA infection, themethod comprises: administering, to a subject who has nosocomialinfection, Staphylococcus infection, or MRSA infection, or a symptom ofnosocomial infection or MRSA infection, or is at risk of nosocomialinfection, Staphylococcus infection, or MRSA infection, an effectiveamount of soluble chitosan or derivatized chitosan, thereby treating orpreventing the nosocomial infection, Staphylococcus infection, or MRSAinfection, reducing or preventing the spread of nosocomial infection,Staphylococcus infection, or MRSA infection, reducing Staphylococcus orMRSA load, or treating or preventing the symptom of nosocomialinfection, Staphylococcus infection, or MRSA infection.
 2. The method ofclaim 1, wherein the soluble chitosan or derivatized chitosan isadministered to treat nosocomial infection.
 3. The method of claim 1,wherein the soluble chitosan or derivatized chitosan is administered totreat MRSA infection. 4-14. (canceled)
 15. The method of claim 1,wherein the subject is a human or an animal.
 16. The method of claim 1,wherein the nosocomial infection or MRSA infection has previously beentreated with an antibiotic without a soluble chitosan or derivatizedchitosan.
 17. The method of claim 1, wherein the subject has one or moresymptoms selected from a group consisting of: a red, swollen and painfularea on the skin, drainage of pus or other fluids from the infectedsite, fever, skin abscess, warmth around the infected area, chest pain,chills, fatigue, fever, malaise, headache, muscle aches, rash, wound,skin breach, and/or shortness of breath.
 18. (canceled)
 19. The methodof claim 1, wherein the soluble chitosan or derivatized chitosan isadministered topically, enterally or parenterally.
 20. The method ofclaim 1, wherein the soluble chitosan or derivatized chitosan isadministered by inhalation spray.
 21. (canceled)
 22. The method of claim1, wherein the soluble chitosan or derivatized chitosan is notadministered in combination with a second therapy.
 23. The method ofclaim 1, wherein the MRSA is selected from EMRSA15 strain, EMRSA16strain, CC8 strain designated ST8:USA300, ST8:USA400 strain, ST8:USA500strain, ST59:USA1000 strain, ST59 strain, ST80 strain, ST93 strain, MW-2strain, MNHO strain, or clinical isolates from a hospital. 24.(canceled)
 25. The method of claim 1, wherein the soluble chitosan orderivatized chitosan reduces MRSA load in the subject by at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%, compared to theMRSA load in the subject before treatment with the soluble chitosan orderivatized chitosan.
 26. The method of claim 1, wherein the solublechitosan is soluble in aqueous solution between pH 6.8 and pH 7.4. 27.The method of claim 1, wherein the soluble chitosan has a moleculeweight less than 10,000 kDa.
 28. The method of claim 1, wherein thederivatized chitosan comprises a chitosan of the following formula (I):

wherein: n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl, and agroup of formula (II):

or R¹, when taken together with the nitrogen to which it is attached,forms a guanidine moiety, wherein R² is hydrogen or amino; and R³ isamino, guanidino, C₁-C₆ alkyl substituted with an amino or guanidinomoiety, or a natural or unnatural amino acid side chain, wherein atleast 25% of R¹ substituents are H, at least 1% of R¹ substituents areacetyl, and at least 2% of R¹ substituents are a group of formula (II).29. The method of claim 28, wherein between 25-95% of R¹ substituentsare hydrogen.
 30. The method of claim 28, wherein between 55-90% of R¹substituents are hydrogen.
 31. The method of claim 28, wherein between1-50% of R¹ substituents are acetyl.
 32. The method of claim 28, whereinbetween 4-20% of R¹ substituents are acetyl.
 33. The method of claim 28,wherein between 2-50% of R¹ substituents are a group of formula (II).34. The method of claim 28, wherein between 4-30% of R¹ substituents area group of formula (II).
 35. The method of claim 28, wherein 55-90% ofR¹ substituents are hydrogen, 4-20% of R¹ substituents are acetyl, 4-30%of R¹ substituents are a group of formula (II).
 36. The method of claim28, wherein R² is amino and R³ is an arginine side chain.
 37. The methodof claim 28, wherein R¹ is selected from one of the following:

38-53. (canceled)
 54. The method of claim 28, wherein R² is amino thatis substituted with a nitrogen protecting group.
 55. The method of claim28, wherein the nitrogen protecting group is tert-butyloxycarbonyl(Boc). 56-65. (canceled)
 66. The method of claim 28, wherein R³ is C₁-C₆alkyl substituted with a guanidino group.
 67. The method of claim 28,wherein R³ is C₁ alkyl substituted with a guanidino group.
 68. Themethod of claim 28, wherein R³ is C₂ alkyl substituted with a guanidinogroup.
 69. The method of claim 28, wherein R³ is C₃ alkyl substitutedwith a guanidino group.
 70. The method of claim 28, wherein R¹ isselected from one of the following:


71. The method of claim 28, wherein at least 25% of R¹ substituents areH, at least 1% of R¹ substituents are acetyl, and at least 2% of R¹substituents independently selected from any of the formulaespecifically shown above.
 72. (canceled)
 73. The method of claim 28,wherein the molecular weight of the derivatized chitosan is between5,000 and 1,000,000 Da.
 74. The method of claim 28, wherein themolecular weight of the derivatized chitosan is between 5,000 and350,000 Da.
 75. The method of claim 28, wherein the molecular weight ofthe derivatized chitosan is between 5,000 and 60,000 Da.
 76. The methodof claim 28, wherein the molecular weight of the derivatized chitosan isbetween 5,000 and 35,000 Da.
 77. The method of claim 28, wherein thederivatized chitosan is soluble in aqueous solution between pH 6.8 andpH 7.4.
 78. The method of claim 28, wherein the derivatized chitosan issubstantially free of other impurities. 79-295. (canceled)